Time-resolved Laue crystallography, as implied by its name, can only be performed on crystalline samples. The intermolecular forces that maintain crystalline order constrain large amplitude conformational motion, and this loss of flexibility may perturb or even inhibit the function of a protein. Nonetheless, Laue crystallography stands alone in its ability to acquire near-atomic structural information on ultrafast time scales. On the other hand, X-rays can also extract structural information from molecules in solution where the full range of conformational motion is permitted. Because there is no long-range order in protein solutions, Small- and Wide-Angle-X-ray-Scattering (SAXS/WAXS) from the protein is diffuse. However, diffraction rings in the SAXS/WAXS pattern are influenced by the size and shape of the protein. Though this structural information is not at atomic resolution, it does provide a fingerprint that can be correlated via models with the protein structure. Time-dependent changes of the WAXS fingerprint can therefore be used to assess which models best describe the reaction pathway in solution. When we first set out to study the quaternary structure transition of hemoglobin with this technique, we observed a substantial change of the WAXS spectrum at the earliest time we could measure, which suggested that the WAXS spectrum is sensitive to tertiary structure changes as well as quaternary structure changes. To prove this point, we recorded the WAXS spectrum of photolyzed carbon monoxymyoglobin (MbCO) and observed a sizable change in the WAXS pattern. According to X-ray structures of MbCO and deoxy Mb, the rms difference in their atomic positions is less than 0.5 Angstrom. This unexpected ability to sense such small but systematic structure changes is quite encouraging, and has spurred us to continue our efforts to develop this new experimental methodology. Our efforts to characterize the quaternary structure transition by time-resolved WAXS has proven more challenging than envisaged. One of the problems faced in these studies is the substantial geminate ligand rebinding that follows laser photolysis. Even though we can photolyze the majority of the bound ligands with a 2-ns pump pulse, more than 30% of them rebind after the pump pulse is over, and a heterogeneous mixture of ligation states are formed. Some of these states are capable of undergoing the R->T quaternary structure transition, but at different rates. For example, it has been reported that Hb(CO)2 can undergo a quaternary structure transition, provided the two ligands are not both bound to alpha chains or both bound to beta chains. One would expect HbCO to undergo the transition at a faster rate, and Hb (no bound ligands) would be fastest. To produce a more homogeneous distribution of ligation states, we sacrificed our time resolution by photolyzing the Hb(CO)4 with a 100-150 ns duration laser pulse. Because the pump pulse was long compared to the 33 ns geminate rebinding time, the laser pulse had multiple opportunities to drive CO away from the primary docking site and thereby generated a high population of Hb with no CO bound. Samples for these studies were prepared at a relatively high concentration (2 mM) to maximize the protein scattering signal. At this concentration, complete photolysis produces a CO concentration in solution of 9 mM, which is 9-fold higher that the equilibrium concentration of CO in the solution (when equilibrated with 1 atm CO). The excess CO leads to rapid bimolecular rebinding to the hemes, which ultimately limits the fractional population that can attain the T state. Though our aim was to continue these Hb studies at a lower protein concentration, our efforts were thwarted by a lack of beamtime at the ESRF. Thus, we have invested much effort over the past year to develop the infrastructure required to pursue time-resolved X-ray scattering studies at the APS. Significantly, we have succeeded in extending our time-resolved capabilities into the SAXS region while expanding even further the accessible WAXS range. Moreover, we have developed a sample translation stage capable of 5G acceleration and can move the sample to a new position between X-ray pulses at a repetition frequency as high as 41 Hz. The S/N of the data acquired with this infrastructure is significantly improved beyond that attained earlier at the ESRF. The X-ray scattering signal is dominated by scatter from sources other than the protein, and the signal ascribed to time-resolved structural changes is 3 to 4 orders of magnitude weaker than the static scattering signature. We have found that instabilities of the X-ray source generate signals that can be much larger than the pump-induced structural change. To isolate the signatures of various contributions to the scattering signal, we have developed sophisticated computational methods to analyze the scattering patterns, some of which employ singular value decomposition (SVD). The feedback from this analysis has led to a major redesign of the sample holder and the data acquisition protocol. Preliminary results suggest that we should soon be capable of extracting the protein contribution to the total scattering signal with near shot-noise-limited detection sensitivity. Our data analysis demonstrated the hypersensitivity of the water scattering to temperature: temperature changes of less than one degree produce a signal that is large compared to the signal due to protein structural change. We aim to exploit this temperature sensitivity and extract time-resolved calorimetric information alongside the time-resolved structural characterization.